Project Summary/Abstract The central goal of this project is to understand the role of BRD4 as an epigenetic vulnerability in acute myeloid leukemia. BRD4 is a bromodomain-containing reader of acetylated transcription factors and histones, and is a founding member of an emerging class of anti-cancer drug targets that function as transcriptional coactivators. While early stage clinical trials have revealed activity of BRD4 inhibitors in relapse-refractory AML patients, several challenges remain to successfully implement BRD4 inhibitors in the clinic. Following our successful effort over the past four years in revealing factors that function upstream and downstream of BRD4 to support AML maintenance, we now propose to address three fundamental gaps in our understanding of BRD4 as an epigenetic vulnerability in this disease. First, we seek to address one of the most perplexing issues underlying BRD4 as a therapeutic target, which is related to the source of specificity of transcriptional effects of BRD4 inhibitors. In Aim 1, we will test the hypothesis that the leukemogenic transcription factor MYB uses BRD4 as a coactivator to maintain the enhancer landscape in leukemia cells. The MYB-BRD4 interaction will be defined on a biochemical and genetic level, and will determine whether the linkage between these two regulators forms the basis for the gene-specific effects and the therapeutic index of BRD4 inhibitors in this disease. A second area of focus will be the Mediator complex, which is a 30-subunit protein complex that we have recently demonstrated is tethered to the genome by BRD4 at specific cis-elements. The intimate linkage between BRD4 and Mediator, together with the rich diversity of protein surfaces within this super-complex, leads us to evaluate in Aim 2 of this proposal whether discrete protein modules within Mediator might be required for AML maintenance, but dispensable for normal biology. This Aim will leverage the CRIPSR exon-scanning technique, which we recently developed to probe the essentiality of protein domains in sustaining cancer. We anticipate that a new generation of BRD4-like vulnerabilities will be present within this complex, and will be candidates for drug discovery. Finally, we seek to understand and overcome acquired resistance to BRD4 inhibition. Our domain-focused CRISPR screens have nominated the lysine deacetylase SIRT6 and lysine methyltransferase SUV420H2 as factors that modulate the sensitivity of AML cells to BRD4 inhibitors. In addition, we have identified LSD1 inhibition as a strategy for overcoming BRD4i resistance. In Aim 3, we will determine the function of these regulators in AML and their role in mediating the anti-leukemia response of BRD4 inhibition. This avenue of research may expose predictive biomarkers of BRD4 inhibitor responses, and potential avenues for overcoming resistance in the clinic. This research will continue to reveal principles of epigenetic perturbations in a therapeutic context, which can guide the development of next-generation therapeutics within this target class in oncology.